Takemi Enomoto

Changes in the level of poly ADP-ribosylation during a cell cycle

Abstract In order to analyze the fluctuation of the poly ADP-ribosylation level during the cell cycle of synchronously growing He La S3 cells, we have developed three different assay systems; intact and disrupted nuclear systems, and poly(ADP-ribose) polymerase in vitro system. The optimum conditions for poly ADP-ribosylation in each assay system were similar except the pH optimum. Under the conditions favoring poly ADP-ribosylation, little radioactivity incorporated into poly(ADP-ribose) was lost after termination of the poly ADP-ribosylation by addition of nicotinamide which inhibits the reactions by more than 90% in any system. In the intact nuclear system, the level of poly ADP-ribosylation increased slightly subsequent to late G2 phase with a peak at M phase. The high level of poly ADP-ribosylation in M phase was also confirmed by using selectively collected mitotic cells which were arrested in M phase by Colcemid. The level in mitotic chromosomes was 5.1-fold higher than that in the nuclei from logarithmically growing cells. Colcemid has no effect on the poly ADP-ribosylation. In the disrupted nuclear system, a relatively high level of poly ADP-ribosylation was observed during mid S-G2 phase. When poly(ADP-ribose) polymerase was extracted from the nuclei with a buffer solution containing 0.3 M KCl, more than 90% of the enzyme activity was recovered. The poly(ADP-ribose) polymerase in vitro system was dependent on both DNA and histone—10 μg each. In the enzyme system, enzyme activity was detected throughout the cell cycle and was observed to be highest in G2 phase. The high level at M phase observed in the intact nuclear system was not seen in the other two systems. Under the assay conditions, little influence of poly(ADP-ribose) degrading enzymes was noted on the level of poly ADP-ribosylation in any of the three systems. This was confirmed at various stages during the cell cycle through pulse-labeling and “chasing” by adding nicotinamide.

Distribution of poly(ADP-ribose) in histones of HeLa cell nuclei.

Abstract The reaction product obtained from HeLa cell nuclei incubated with [ 3 H]NAD was specifically hydrolyzed with snake venom phosphodiesterase. Analysis of the hydrolyzed product revealed that it is a homopolymer consisting of 4–5 repetition of ADP-ribose units. The [ 3 H]poly ADP-ribosylated histone fraction was anslyzed by urea-acetic acid polyacrylamide gel electrophoresis. The radioactive peak was clearly separated from the stained histone H1 band, while a slight overlap was observed. When chromatographed on a SP-Sephadex C-50 column, more than 90% of the radioactivity of [ 3 H]poly(ADP-ribose) was eluted in accordance with histones but not with nonhistone contaminants. On a sodium dodecyl sulfate polyacrylamide gel electrophoresis, a major radioactive peak appeared at a position very close to the histone Hl band, which disappeared by the treatment with alkali prior to electrophoresis. A selective extraction of histone Hl with 5% perchloric acid showed that histone Hl contained about 85% of the radioactivity incorporated into whole histones.

The SUMO pathway is required for selective degradation of DNA topoisomerase IIβ induced by a catalytic inhibitor ICRF-1931

DNA topoisomerase I and II have been shown to be modified with a ubiquitin‐like protein SUMO in response to their specific inhibitors called ‘poisons’. These drugs also damage DNA by stabilizing the enzyme–DNA cleavable complex and induce a degradation of the enzymes through the 26S proteasome system. A plausible link between sumoylation and degradation has not yet been elucidated. We demonstrate here that topoisomerase IIβ, but not its isoform IIα, is selectively degraded through proteasome by exposure to the catalytic inhibitor ICRF‐193 which does not damage DNA. The β isoform immunoprecipitated from ICRF‐treated cells was modified by multiple modifiers, SUMO‐2/3, SUMO‐1, and polyubiquitin. When the SUMO conjugating enzyme Ubc9 was conditionally knocked out, the ICRF‐induced degradation of topoisomerase IIβ did not occur, suggesting that the SUMO modification pathway is essential for the degradation.

Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.

Effect of heliquinomycin on the activity of human minichromosome maintenance 4/6/7 helicase.

The antibiotic heliquinomycin, which inhibits cellular DNA replication at a half‐maximal inhibitory concentration (IC50) of 1.4–4 μm, was found to inhibit the DNA helicase activity of the human minichromosome maintenance (MCM) 4/6/7 complex at an IC50 value of 2.4 μm. In contrast, 14 μm heliquinomycin did not inhibit significantly either the DNA helicase activity of the SV40 T antigen and Werner protein or the oligonucleotide displacement activity of human replication protein A. At IC50 values of 25 and 6.5 μm, heliquinomycin inhibited the RNA priming and DNA polymerization activities, respectively, of human DNA polymerase‐α/primase. Thus, of the enzymes studied, the MCM4/6/7 complex was the most sensitive to heliquinomycin; this suggests that MCM helicase is one of the main targets of heliquinomycin in vivo. It was observed that heliquinomycin did not inhibit the ATPase activity of the MCM4/6/7 complex to a great extent in the absence of single‐stranded DNA. In contrast, heliquinomycin at an IC50 value of 5.2 μm inhibited the ATPase activity of the MCM4/6/7 complex in the presence of single‐stranded DNA. This suggests that heliquinomycin interferes with the interaction of the MCM4/6/7 complex with single‐stranded DNA.

Human ubiquitin-activating enzyme (E1): Compensation for heat-labile mouse E1 and its gene localization on the X chromosome

We have constructed interspecific somatic cell hybrids between a temperature-sensitive (ts) mutant cell line of mouse FM3A cells, ts85, that has a heat-labile ubiquitin-activating enzyme (E1) and a human diploid fibroblast cell line, IMR-90. A hybrid clone that could grow stably at a nonpermissive temperature (39 degrees C) was obtained. Segregation of the hybrid cells at a permissive temperature (33 degrees C) gave rise to temperature-sensitive clones. The electrophoresis of extracted histones and karyotype analysis of the segregants revealed a close correlation of the ability to grow at 39 degrees C, the presence of uH2A (ubiquitin-H2A semihistone) at 39 degrees C, and the presence of the human X chromosome. One of the hybrid clones that could grow at the nonpermissive temperature contained the X chromosome as the only human chromosome. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern of affinity-purified E1 showed that this hybrid clone contained both human and mouse type E1. Thus we conclude that the functional gene for human E1 is located on the X chromosome.

Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase α-activity

tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.

DNA-dependent ATPase B of FM3A cells: Its separation from DNA polymerase α

One form of DNA‐dependent ATPase (DNA‐dependent ATPase B) has been purified from FM3A cells. In this report, we describe the association of DNA polymerase α activity with DNA‐dependent ATPase B through a series of purification steps and the final separation of the two enzymes by glycerol gradient centrifugation operated at a low salt concentration.

A system of DNA replication in HeLa nuclei treated with inhibitors of protein synthesis.

An in vitro DNA synthesizing system consisting os isolated nuclei from HeLa cells which had been treated with inhibitors of protein synthesis was investigated. Treatment with both 30 microgram/ml cycloheximide and 10 microgram/ml puromycin of S-phase cells reduced the rate of DNA synthesis immediately; however, the overall DNA synthesis continued for up to 4 h with a diminished rate and then ceased. In the nuclei which were isolated from the cells which had been incubated with these drugs for 6 h, little incorporation of [3H]TTP into acid-insoluble materials was observed. Addition of cytosol prepared from cells actively synthesizing DNA induced the incorporation of [3H]TTP in these nuclei, while little induction was observed by the addition of cytosol prepared from drug-treated cells in spite of the fact that the latter cytosol stimulated DNA synthesis in isolated nuclei from non-treated cells. The induced DNA synthesis was shown to require Mg2+, all four deoxyribonucleoside triphosphates and ATP, and to proceed discontinuously. The activity inducing DNA synthesis in drug-treated nuclei fluctuated with the phases in a cell cycle and it was not ascribed solely to DNA polymerase alpha nor to DNA ligase.

Purification and characterization of mouse DNA polymerase α devoid of primase activity

A simple method was developed for the isolation of primase‐free DNA polymerase‐α from the DNA polymerase‐α‐primase complex of mouse FM3A cells. The polymerase was separated from primase submits by chromatography on a single‐stranded DNA‐cellulose column in the presence of 50% etylene glycol. The primase‐free DNA polymerase‐α contained two polypeptides with molecular masses of 180000 and 68000. Analysis of the DNA products with poly(dA)‐oligo(dT)10 as template‐primer revealed that both primase‐free DNA polymerase‐α and the DNA polymerase‐α‐primase complex predominantly synthesized short DNA with less than 30 nucleotides, but that the DNA polymerase‐α‐primase complex also synthesized some longer DNA with more than 300–400 nucleotides.